Hello to all.
I am performing a thesis on Cognitive disorder modeling in rats and the effect of
Pharmacological Treatment on the disease and the expression of certain miRNAs.
I am seeking guidance from any researcher who has experience in "Rat brain tissue miRNA analysis" to help me understand more about my project so I would be grateful for even the smallest info and tips based on your experience.
I would also be really thankful if anyone could help me with informations about U6 as a housekeeping gene and primer design for this sequence.
Thanks in advance.
hi Pouria Bahmani Kolour
Well, I may not have very specific points for "Rat brain tissue", but widely accepted and feasible to do steps are.
-You can even isolate total RNA using the Trizol method and use Qiagen miScript II RT Kit, which converts all miRNA in a sample to miRNA cDNA and then use those cDNA directly for qPCR to check the miR level itself as well as other GOI.
--Kindly prefer at least 3 endogenous control.
***Never rely on only one endogenous control***
---U6 is miRNA endogenous control but along with it includes two more.
----miRNA primer has universal sequences. The miRDB database provides stem-loop & mature miRNA sequence. Prefer predesigned primers of your interested miRNA from the standard company. Nowadays, Companies do a slight modification or say a technology, which enhances the stability and avoid unwanted binding. Qiagen has LNA tech. based on miRNA primers, it has a readymade, pre-mix(FP+RP) pre-designed primer of most of the miRNAs.Just dissolve and use 1ul for per rxn.
Cheers!!!
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